Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays

Area of Science:

• Genomics and Molecular Biology
• Biotechnology
• Clinical Diagnostics

Background:

• Reference genes are crucial for quantitative assays like droplet digital PCR (ddPCR) in cell therapies.
• Accurate reference gene selection is vital due to significant genomic variations in individuals and diseases like cancer.
• Copy number variations (CNVs) and plasmid loss are critical parameters in genetically engineered cell therapies.

Purpose of the Study:

• To identify reliable reference genes for copy number variation analysis in chimeric antigen receptor T cells.
• To find suitable reference genes for plasmid loss analysis in induced pluripotent stem cells using ddPCR.
• To ensure the accuracy and reproducibility of cell therapy monitoring assays.

Main Methods:

• Utilized The Cancer Genome Atlas (TCGA) to screen candidate reference genes for low copy number variance in cancer.
• Employed ddPCR to measure copy numbers of potential reference genes in healthy subjects, cancer cell lines, and various cancer patient samples.
• Evaluated four candidate genes: AGO1, AP3B1, MKL2, and rPP30.

Main Results:

• None of the four evaluated gene regions (AGO1, AP3B1, MKL2, rPP30) were universally amplified or deleted across all relevant cancer types.
• Copy numbers of AP3B1, AGO1, rPP30, and MKL2 were similar in cells from healthy subjects.
• AP3B1, MKL2, and rPP30 demonstrated stable copy numbers across analyzed cancer patient samples, unlike AGO1 which showed alterations.

Conclusions:

• AP3B1, MKL2, and rPP30 are validated as suitable reference genes for ddPCR assays measuring vector copy number in chimeric antigen receptor T cells.
• These genes are appropriate for analyzing cell therapies in patients with acute leukemia, lymphoma, multiple myeloma, and HPV-associated cancers.
• Ongoing evaluation of AGO1 will continue for future sample analyses.